Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery

Abstract

Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea (Aeropyrum pernix and Pyrobaculum calidifontis) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens, and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been 'domesticated', that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA.

Fig. 1. Archaeal conjugative pili.

a, d Representative cryo-electron micrographs of A. pernix and P. calidifontis pili, respectively, from >8000 images collected for each. Scale bars, 50 nm. b, e Side and top views of the A. pernix (b, yellow) and P. calidifontis (e, blue) cryo-EM density maps at resolutions of 3.3 Å and 4.0 Å, respectively. The front half of the filament has been removed in the side views, so that lumens are visible. c, f Atomic models in ribbon representation of the A. pernix (c, yellow) and P. calidifontis (f, blue) pili docked within their respective transparent cryo-EM density maps. The asymmetric unit of the A. pernix pilin CedA1 (c, bright yellow) shows two bound lipids (magenta and blue), while a single asymmetric unit of the P. calidifontis pilin TedC (f, dark blue) shows one bound tetraether lipid (orange). g Genomic loci encompassing the Ted system in different members of the order Thermoproteales. Each of the five genera within the Thermoproteales (genera Caldivirga, Pyrobaculum, Thermocladium, Thermoproteus, and Vulcanisaeta) is represented. Genes encoding the conserved VirB2/CedA1-like pilin protein TedC, VirB6/CedA-like transmembrane channel TedA, and VirB4/CedB-like AAA + ATPase TedB are shown as red, green, and cyan arrows, respectively. Additional conserved genes are also indicated, including VirC1/ParA-like, Rad50, and HerA-like helicase which are shown as orange, magenta, and dark blue arrows, respectively. Other genes are shown in gray. Genomic loci are aligned using the TedC gene and indicated with the corresponding UniProt accession numbers, followed by the organism name. In some species, the components of the Ted systems are encoded within distal genomic loci which are separated from the TedC-encoding loci by dashed lines, and the corresponding genes are identified with their UniProt accession numbers.

Fig. 2. Intricate pilin-lipid interaction networks within archaeal conjugative pili.

a Front and back views of a single asymmetric unit of the P. calidifontis pilus (blue) containing one pilin (TedC) and one phospholipid GDGT-0 (orange). One polar headgroup of the tetraether lipid faces the lumen and the other faces the outside of the pilus. A top view of a subunit, looking down the helical axis, is shown on the left for orientation. The isoprenoid chains of the GDGT-0 lipids are buried between hydrophobic helices and interact closely with helix α1 of the pilin shown but will also interact with α2 of neighboring pilins. b An atomic model of the cyclic GDGT-0 lipid docked within the lipid density. c Lipid density from P. calidifontis (blue). The density is very well resolved and shows that the GDGT-0 lipids have one head group facing the outside of the filament and the other head group facing the lumen. d A single pilin (blue) contacts four surrounding GDGT-0 lipids (orange). e The front and back view of the CedA1 pilin (yellow) of A. pernix. The asymmetric unit contains two lipids and one pilin, with the lipid in two different conformations: one having a partially folded shape (blue) and the other a crescent-like shape (magenta). The crescent head group is facing the outside of the pilus while the isoprenoid chains are buried between the pilin subunits. The partially folded lipid’s phosphate head group is facing the lumen of the pilus and the isoprenoid chains are bent and buried between the subunits. A top view of a subunit, looking down the helical axis, is shown on the left for orientation. For both lipids the contacts with the pilin are mediated by hydrophobic residues such as leucine, isoleucine and valine from helices α1 and α2. The crescent-like lipid has one hydrophobic interaction with the partially folded lipid. f Atomic models for the crescent-shaped and partially folded lipid docked into the lipid densities. g Lipid density from A. pernix (yellow). The lipid density is less resolved than in P. calidifontis but shows two C25-C25-diether lipids, one of which forms a crescent-like shape (arrow 1) and the other forms a partially folded shape (arrow 2). Both lipids are capped with extra density (arrow 3) which is likely a dihexose sugar attached to the phosphate head group. h A single protein subunit (yellow) makes contacts with ten lipids (five crescent-shaped (pink), and five partially folded (blue).

Fig. 3. Structure of the A. tumefaciens T-pilus.

a Representative cryo-electron micrograph of A. tumefaciens T-pilus, from 8,363 images collected. Scale bar, 50 nm. b Side and top view of the T-pilus cryo-EM density map at a resolution of 3.5 Å. The front half of the filament has been removed in the side view, and we are looking at the lumen. c While there is a 1:1 stoichiometry of lipids to pilins in the filament, a pilin (red) makes contact with four lipids (green). d Atomic model of the T-pilus in ribbon representation docked within the transparent cryo-EM density map. A single subunit model is shown in red. An inset on the right shows front and back views of the VirB2 subunit.

Fig. 4. Comparison of archaeal and bacterial conjugative pili.

a Sequence alignments of archaeal (top) and bacterial (bottom) mating pili. Sequences of mature pilins are shown with the secondary structure elements determined from the structure for the pilins shown above and below the corresponding sequences. Kinks in the α-helices are indicated with triangles. b, e Comparison of the lumen and outer diameter of archaeal P. calidifontis (blue) and A. pernix (yellow) with bacterial pili: A. tumefaciens T-pilus (red), pED208 (mauve), F-pilus (purple) and pKpQIL (green). The outer diameters range between 74 Å to 87 Å. The lumen diameters range between 16 Å to 26 Å, but in some cases cannot be easily reduced to a single number. c, d Atomic models with a single strand, shown in gray, show connectivity between the subunits. This connectivity is also represented with helical nets for the archaeal and bacterial pili. The helical nets show the unrolled surface lattice viewed from the outside of the filament. Each point represents a subunit, and the dotted lines are drawn to highlight the fact that all have right-handed 5-start helices. All of the pili have substantial connectivity between subunits along these 5-start helices.

Fig. 5. Proposed conjugation mechanisms between donor and recipient cells in archaea (left) and bacteria (right).

The schematic shows how ssDNA substrates are generated by the HerA-NurA machinery in the donor archaeal cells and by the plasmid-encoded relaxosome in bacteria. Note that CedA and, potentially, Ted systems function as DNA importers rather than DNA exporters, contrary to the bacterial plasmid conjugative machinery.